The present invention generally relates to methods and compositions for the treatment of mammals, including humans, following an ischemic or traumatic injury to the central nervous system.
Numerous proteins have now been identified and characterized as morphogenetic or growth factors, regulating cell proliferation and/or differentiation of tissues in vertebrates, including mammals. Typically these growth factors exert their effects on specific subsets of cells and/or tissues. Thus, for example, epidermal growth factors, nerve growth factors, fibroblast growth factors, various hormones, and many other proteins inducing or inhibiting cell proliferation or differentiation have been identified and shown to affect some subset of cells or tissues.
Neurotrophic factors are polypeptides that are required for the development of the nervous system. The first neurotrophic factor discovered, nerve growth factor (NGF), is now known to be a part of a large family of growth factors, which also includes BDNF, NT3, and NT4/NT5. The dimeric proteins defined in PCT Publication No. WO 94/03200 as morphogens constitute another family of proteins believed to play an important role in neural development (Jones, et al. (1991) Development 111: 531-542; Ozkaynak, et al. (1992) J. Biol. Chem. 267: 25220-25227; Lein, et al. (1995) Neuron 15: 597-605).
These proteins, referred to herein as xe2x80x9cmorphogenic proteinsxe2x80x9d or xe2x80x9cmorphogens,xe2x80x9d are competent to act as true tissue morphogens, able, on their own, to induce the proliferation and differentiation of progenitor cells into functional mammalian body tissue. The proteins include members of the family of bone morphogenetic proteins (BMPs) which were initially identified by their ability to induce ectopic, endochondral bone morphogenesis.
Morphogens generally are classified in the art as a subgroup of the TGF-xcex2 superfamily of growth factors (Hogan (1996) Genes and Development 10: 1580-1594). Members of the morphogen family of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF-3 (also known as Vgr2), GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), the Xenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, and NEURAL. The members of this family encode secreted polypeptide chains sharing common structural features, including processing from a precursor xe2x80x9cpro-formxe2x80x9d to yield a mature polypeptide chain competent to dimerize, and containing a carboxy terminal active domain of approximately 97-106 amino acids. All members share a conserved pattern of cysteines in this domain and the active form of these proteins can be either a disulfide-bonded homodimer of a single family member, or a heterodimer of two different members (see, e.g., Massague (1990) Annu. Rev. Cell Biol. 6: 597; Sampath, et al. (1990) J. Biol. Chem. 265: 13198). See also, U.S. Pat. No. 5,011,691; U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085-2093, Wharton et al. (1991) PNAS 88: 9214-9218), (Ozkaynak (1992) J. Biol. Chem. 267: 25220-25227 and U.S. Pat. No. 5,266,683); (Celeste etal. (1991) PNAS 87: 9843-9847); (Lyons et al. (1989) PNAS 86: 4554-4558). These disclosures describe the amino acid and DNA sequences, as well as the chemical and physical characteristics of these morphogenic proteins. See also Wozney et al. (1988) Science 242: 1528-1534); BMP-9 (WO 93/00432, published Jan. 7, 1993); DPP (Padgett et al. (1987) Nature 325: 81-84; and Vg-1 (Weeks (1987) Cell 51: 861-867).
Morphogens are expressed naturally in a variety of tissues during development, including those of the developing nervous system (Ozkaynak, et al. (1990) EMBO J. 9: 2085-2093; Ozkaynak, et al. (1991) Biochem. Biophys. Res. Commun. 179:116-123; Ozkaynak, et al. (1992) supra).
Vascular diseases of the nervous system rank first in frequency amongst all the neurologic diseases; they constitute about fifty percent of all neurologic hospital admissions to adult wards. The cardinal feature of cerebrovascular disease is the stroke, a term that connotes the sudden and dramatic development of a focal neurologic deficit. Obstruction of a nutrient artery supplying a locus of the central nervous system by, for example, a thrombus or an embolus or a failure of the systemic circulation and hypotension, if severe and prolonged enough, can deprive brain tissue of blood and oxygen, leading to disruption of physiologic function, subsequent death of neurons, and necrosis (infarction) of the affected locus. In hemorrhagic infarction, an extravasation of blood occurs into the brain tissue, the subarachnoid space, or both. Damage results from physical disruption of the region directly involved and pressure of the mass of blood on the surrounding tissue.
The neurologic deficit in a stroke reflects both the location and the size of the infarct or hemorrhage in the brain. Hemiplegia is the classic sign of vascular disease and occurs with strokes involving the cerebral hemisphere or the brainstem. However, depending on its location, a stroke may also give rise to many other manifestations accompanying or independent of hemiplegia, including numbness, sensory deficit, dysphasia, blindness, diplopia, dizziness, and dysarthria.
Patients who suffer a xe2x80x9cstroke,xe2x80x9d or any other form of cerebral ischemic or traumatic injury, usually recover partially, but often remain mildly to severely debilitated. For example, total infarction of the middle cerebral artery in a human results in a contralateral hemiplegia, hemianesthesia, homonymous hemianopia, global or total sensorimotor aphasia (left hemisphere), and apractagnosia (right hemisphere). Once established, the motor, sensory, and language deficits usually remain static or very little improved after the passage of months or even years. Seldom can the patient ever again communicate effectively. Currently, aside from physical therapy, there is no treatment that reliably improves the prognosis of a patient who has suffered a stroke or any similar injury of the central nervous system.
The present invention is directed to methods and compositions for treatment of mammals afflicted with an ischemic or traumatic injury of the central nervous system. In particular, the invention provides treatments for mammals in whom central nervous system tissue has been damaged or lost due to stroke or a similar disruption in blood flow, or due to infliction of physical (e.g., mechanical) trauma affecting the central nervous system. The methods and compositions provided herein capitalize upon the discovery that administration of a morphogen to such a mammal provides significant improvement in central nervous system function, even when administered after central nervous system tissue has been damaged. The methods involve the administration of dimeric proteins defined as morphogens, inducers of these morphogens, or agonists of the corresponding morphogen receptors, or implantation of cells stimulated by exposure to the morphogens.
Accordingly, the invention features a method for treating a mammal who has suffered an injury to the central nervous system, such as a stroke or a traumatic injury. The method involves administering an effective dose of morphogen to the mammal at least six hours after the onset of the injury; for example, twelve, twenty-four, or forty-eight hours or even longer following the onset of injury.
The treatment regimen according to the invention is carried out in terms of administration mode, timing of the administration, and dosage, so that the functional recovery from impairment of the central nervous system is enhanced. The compositions of the present invention will contain therapeutically-effective amounts of the morphogen, morphogen inducers or agonists of morphogen receptors. That is, the compositions will contain an amount which provides appropriate concentrations of the agent to the affected nervous system tissue for a time sufficient to stimulate a detectable restoration of central nervous system function, up to and including a complete restoration thereof. The effective amount of morphogen can be provided in a single administration, in two administrations or in a plurality of administrations. Where the effective amount of morphogen is provided in a plurality of administrations, the morphogen is preferably administered to the mammal daily. In an alternative preferred embodiment, the morphogen is administered to the mammal biweekly (e.g., every three or four days). In a further alternative preferred embodiment, the morphogen is administered to the mammal once a week.
Practice of the invention confers significant clinical benefit on the afflicted mammal, in that the invention beneficially confers clinically relevant improvement in at least one of the mammal""s motor coordination functions (e.g., posture, balance, grasp, gait), sensory perceptions (e.g., vision, touch, taste, olfaction, proprioception), or speech. Clinically relevant improvement can range from a detectable improvement to a complete restoration of an impaired or lost central nervous function.
The invention can be used to treat adverse consequences of central nervous system injuries that result from a variety of conditions. Thrombus, embolus, and systemic hypotension are among the most common causes of stroke. Other injuries may be caused by hypertension, hypertensive cerebral vascular disease, rupture of an aneurysm, an angioma, blood dyscrasia, cardiac failure, cardiac arrest, cardiogenic shock, kidney failure, septic shock, head trauma, spinal cord trauma, seizure, bleeding from a tumor, or other loss of blood volume and/or pressure. Administration of a morphogen according to the invention confers significant clinical benefit, even when administration occurs a significant amount of time following the injury.
Generally, the morphogens useful in the methods and compositions of the invention are dimeric proteins that induce morphogenesis of one or more eukaryotic (e.g., mammalian) cells, tissues or organs. Of particular interest herein are morphogens that induce morphogenesis at least of bone or neural tissue. Morphogens comprise a pair of polypeptides that, when folded, adopt a configuration sufficient for the resulting dimeric protein to elicit morphogenetic responses in cells and tissues displaying receptors specific for said morphogen. That is, the morphogens generally induce a cascade of events including all of the following in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells. xe2x80x9cProgenitorxe2x80x9d cells are uncommitted cells that are competent to differentiate into one or more specific types of differentiated cells, depending on their genomic repertoire and the tissue specificity of the permissive environment in which morphogenesis is induced. Morphogens further can delay or mitigate the onset of senescence- or quiescence-associated loss of phenotype and/or tissue function. Morphogens still further can stimulate phenotypic expression of differentiated cells, including expression of metabolic and/or functional, e.g., secretory, properties thereof. In addition, morphogens can induce redifferentiation of committed cells under appropriate environmental conditions. As noted above, morphogens that induce proliferation and/or differentiation at least of neural tissue, and/or support the growth, maintenance and/or functional properties of neural tissue, are of particular interest herein. See, for example, WO 92/15323, WO 93/04692 and WO 94/03200 for more detailed disclosures as to the tissue morphogenic properties of these proteins.
As used herein, the terms xe2x80x9cmorphogen,xe2x80x9d xe2x80x9cbone morphogen,xe2x80x9d xe2x80x9cbone morphogenic protein,xe2x80x9d xe2x80x9cBMP,xe2x80x9d xe2x80x9cmorphogenic proteinxe2x80x9d and xe2x80x9cmorphogenetic proteinxe2x80x9d all embrace the class of proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 are provided in SEQ ID NOs: 4 and 5, respectively. For ease of description, hOP-1 is recited herein below as a representative osteogenic protein. It will be appreciated by the artisan of ordinary skill in the art, however, that OP-1 merely is representative of the TGF-xcex2 subclass of true tissue morphogens competent to act as morphogenic proteins, and is not intended to limit the description. Other known, and useful proteins include, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, NODAL, UNIVIN, SCREW, ADMP, NEURAL and morphogenically active amino acid variants thereof. Thus, in one embodiment, preferred morphogenic proteins, include but are not limited to, OP-1, OP-2, BMP-2, BMP-4, BMP-5, and BMP-6. In addition, as will be appreciated by the artisan of ordinary skill in the art any one of the morphogenic proteins recited herein also could be used as a reference sequence.
In another preferred embodiment, the proteins useful in the invention include biologically active species (phylogenetic) variants of any of the morphogenic proteins recited herein, including conservative amino acid sequence variants, proteins encoded by degenerate nucleotide sequence variants, and morphogenically active proteins sharing the conserved seven cysteine skeleton as defined herein and encoded by a DNA sequence competent to hybridize under standard stringency conditions to a DNA sequence encoding a morphogenic protein disclosed herein, including, without limitation, OP-1 and BMP-2 or BMP4. In still another embodiment, useful morphogens include those sharing the conserved seven cysteine domain and sharing at least 70% amino acid sequence homology (similarity) within the C-terminal active domain of a reference morphogen sequence, as defined herein below. In a preferred embodiment, the reference sequence is OP-1.
In still another embodiment, the morphogens useful in the methods and compositions of the invention can be defined as morphogenically active proteins having any one of the generic sequences defined herein, including OPX and Generic Sequences 7 and 8 (SEQ ID NOs: 1 and 2 respectively), or Generic Sequences 9 and 10 (SEQ ID NOs: 6 and 7, respectively). OPX accommodates the homologies between the various species of the osteogenic OP-1 and OP-2 proteins, and is described by the amino acid sequence presented herein below and in SEQ ID NO: 3. Generic sequence 9 is a 96 amino acid sequence containing the six cysteine skeleton defined by hOP-1 (residues 335-431 of SEQ ID NO: 5) and wherein the remaining residues accommodate the homologies of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, UNIVIN, NODAL, DORSALIN, NEURAL, SCREW and ADMP. That is, each of the non-cysteine residues is independently selected from the corresponding residue in this recited group of proteins. Generic Sequence 10 is a 102 amino acid sequence which includes a 5 amino acid sequence added to the N-terminus of the Generic Sequence 9 and defines the seven cysteine skeleton defined by hOP-1 (330-431 SEQ ID NO: 5). Generic Sequences 7 and 8 are 96 and 102 amino acid sequences, respectively, containing either the six cysteine skeleton (Generic Sequence 7) or the seven cysteine skeleton (Generic Sequence 8) defined by hOP-1 and wherein the remaining residues non-cysteine accommodate the homologies of: OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1, and GDF-1.
As contemplated herein, the family of morphogenic proteins described herein includes longer forms of a given protein, as well as phylogenetic, e.g., species and allelic variants, and biosynthetic mutants, including C-terminal addition and deletion mutants and variants, such as those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration still allows the protein to form a dimeric species having a conformation capable of inducing neural tissue formation in a mammal when provided to a morphogenically permissive site in a mammal. In addition, the morphogenic proteins useful in the invention may include forms having varying glycosylation patterns and varying N-termini, may be naturally occurring or biosynthetically derived, and may be produced by expression of recombinant DNA in prokaryotic or eucaryotic host cells. The proteins are active as a single species (e.g., as homodimers, including chimeras), or combined as a mixed species, including heterodimers.
Of particular interest herein are morphogens which, when provided to neural tissue of a mammal, induce or maintain the normal state of differentiation and growth of that tissue. In a currently preferred demonstrative embodiment, the present morphogens induce or reinduce a developmental cascade of cellular and molecular events that culminates in the formation of vertebrate central nervous system tissue. In other preferred demonstrative embodiments, the present morphogens similarly induce the formation of other vertebrate (e.g., avian or mammalian) body tissues, such as but not limited to bone, cartilage, bone marrow, ligament, tooth dentin, periodontium, liver, kidney, lung, heart or gastrointestinal lining. The present demonstrations can be carried out in the context of developing, embryonic tissue, or at an aseptic, unscarred wound site in post-embryonic tissue. Particularly preferred morphogens induce or trigger a pattern formation cascade in a developing mammalian or avian embryo that culminates in the formation of one or more functionally integrated elements of central or peripheral nervous system. Such morphogens can be used to treat a mammal afflicted with ischemic or traumatic injury of the central nervous system.
The present invention alternatively can be practiced with methods and compositions comprising a morphogen inducer in lieu of a morphogen. A xe2x80x9cmorphogen inducerxe2x80x9d is a compound that stimulates the in vivo production (i.e., transcription, translation, and/or secretion) of a therapeutically-effective concentration of an endogenous morphogen in the body of a mammal. An xe2x80x9ceffectivexe2x80x9d concentration is sufficient to promote the regeneration or maintenance of neural tissue and/or to inhibit additional loss thereof. Such compounds are understood to include substances which, when administered to a mammal, act on cells that normally are competent to produce and/or secrete a morphogen encoded within the genome of the mammal, and which cause the endogenous level of the morphogen to be increased. Endogenous or administered morphogens can act as endocrine, paracrine or autocrine factors. That is, endogenous morphogens can be synthesized by the cells in which the morphogenetic responses are induced, by neighboring cells, or by cells of a distant tissue, in which case the secreted endogenous morphogen is transported to the site of morphogenesis, e.g., by the individual""s bloodstream. In preferred embodiments, the inducer stimulates expression and/or secretion of an endogenous morphogen so as to increase amounts thereof available in neural tissue.
In still other embodiments, an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself. An xe2x80x9cagonistxe2x80x9d of a receptor is a compound which binds to the receptor, and for which the result of such binding is similar to the result of binding the natural, endogenous ligand of the receptor. That is, the compound must, upon interaction with the receptor, produce the same or substantially similar transmembrane and/or intracellular effects as the endogenous ligand. Thus, an agonist of a morphogen receptor binds to the receptor and such binding has the same or a functionally similar result as morphogen binding (e.g., induction of morphogenesis). The activity or potency of an agonist can be less than that of the natural ligand, in which case the agonist is said to be a xe2x80x9cpartial agonist,xe2x80x9d or it can be equal to or greater than that of the natural ligand, in which case it is said to be a xe2x80x9cfull agonist.xe2x80x9d Thus, for example, a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen""s receptor may be employed as an equivalent of the morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed. Methods of identifying such agonists are known in the art and include assays for compounds which induce morphogen-mediated responses (e.g, induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation, and the like). Such an agonist may also be referred to as a morphogen xe2x80x9cmimic,xe2x80x9d xe2x80x9cmimetic,xe2x80x9d or xe2x80x9canalog.xe2x80x9d
The morphogens, inducers and agonists of the invention may be administered by any route of administration which is compatible with the selected agent, including by intravenous, subcutaneous, intramuscular, ophthalmic, intraperitoneal, buccal, rectal, vaginal, intraorbital, oral, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracistemal, intracapsular, intranasal or by aerosol administration and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. In addition, various growth factors, hormones, enzymes, therapeutic compositions, antibiotics, or other bioactive agents can be co-administered with the morphogen. Thus, various known growth factors such as NGF, EGF, PDGF, IGF, FGF, TGF-xcex1, and TGF-xcex2, as well as enzymes, enzyme inhibitors and/or chemoattractant/chemotactic factors, can be combined with the morphogen and be delivered to the defect locus.
The method of the invention advantageously stimulates restoration of central nervous system function even when practiced hours, or even days, following an injury to the central nervous system. The invention thus significantly improves on the treatment options available when central nervous system injury occurs and is not diagnosed or treated prior to the death of involved tissue.
The preferred methods, material, and examples that will now be described are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.